1. Homeostatic Control of Memory Cell Progenitors in the Natural Killer Cell Lineage 
Yosuke Kamimura, Lewis L. Lanier 
Cell Reports 
Volume 10, Issue 2, Pages (January 2015)
DOI: /j.celrep
Copyright © 2015 The Authors Terms and Conditions

2. Cell Reports 2015 10, 280-291DOI: (10.1016/j.celrep.2014.12.025)
Copyright © 2015 The Authors Terms and Conditions

3. Figure 1
KLRG1− NK Cells Preferentially Expand and Generate Memory NK Cells
(A and B) KLRG1int+ and KLRG1− NK cells from C57BL/6 mice (CD45.2+) and congenic C57BL/6.SJL mice (CD45.1+), respectively, were sorted using flow cytometry to >99% purity. An equal number (1 × 105 cells) of CD45.2+Ly49H+ KLRG1int+ and CD45.1+Ly49H+ KLRG1− NK cells were mixed and adoptively transferred into Ly49H-deficient hosts (day −1) followed by MCMV infection (day 0). (A) Percentages of CD45.1+ (KLRG1− cells derived) and CD45.1− (KLRG1int+ cells derived) cells in the Ly49H+ NK cell population. (B) Percentages of adoptively transferred CD45.1+ (KLRG1− cells derived) and CD45.2+ (KLRG1int+ cells derived) Ly49H+ NK cells within the total NK cell population.
(C and D) Sorted KLRG1int+ Ly49H+ and KLRG1− Ly49H+ NK cells (1 × 104 of each subset) were adoptively transferred into Ly49H-deficient hosts (day −1) and infected with MCMV (day 0). Virus copy number in peripheral blood (C) and oral lavage (D) was quantitated by real-time qPCR.
Data are representative of three (A and B) and two (C and D) independent experiments. Error bars show SEM (n = 3 for A and B; n = 3–7 for C and D). See also Figure S1.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2015 The Authors Terms and Conditions

4. Figure 2
KLRG1int+ NK Cells Are Increased in T-Cell-Deficient Mice, but Not B-Cell-Deficient Mice
(A) Percentages of KLRG1int+ cells within the total NK cell population in spleen and peripheral blood from the indicated mice.
(B–D) NK cells were isolated from Rag1−/− mice (B), Tcra−/− mice (C), or μMt mice (D) (all CD45.2+) and cotransferred with an equal number of WT (CD45.1+) Ly49H+ NK cells. Graph shows percentages of Ly49H+ cells within the total NK cell population at the indicated time points after MCMV infection.
(E) Percentages of KLRG1int+ cells within the total NK cell population in WT and Rag1−/− mice and in mixed bone marrow chimeric mice (1:1 mixture of WT [CD45.1+] and Rag1−/− mice [CD45.2+] cells). BM, bone marrow.
(F) WT and Rag1−/− NK cells were isolated from the mixed BMC mice (E), adoptively transferred into Ly49H-deficient hosts, and then infected with MCMV. Percentages of WT and Rag1−/− NK Ly49H+ NK cells after MCMV infection are shown.
(G) Percentages of KLRG1int+ cells within the total NK cell population in WT and Rag1−/− mice and in Rag1−/− mice given WT spleen cells (5 × 107 cells).
(H) WT mice, Rag1−/− mice, and the Rag1−/− mice that received WT splenocytes were treated with anti-NK1.1 mAb and rested for 8 weeks. NK cells were isolated from these three groups of mice, and an equal number of Ly49H+ NK cells was adoptively transferred into Ly49H-deficient hosts (day −1) and then infected with MCMV (day 0). Percentages of transferred Ly49H+ NK cells isolated from WT mice, Rag1−/− mice, and in Rag1−/− mice given WT spleen cells after MCMV infection are shown.
(I and J) KLRG1− NK cells were sorted from WT (CD45.1+), Rag1−/− (CD45.2+), and Tcra−/− (CD45.2+) mice, and an equal number of KLRG1−Ly49H+ NK cells from Rag1−/− (CD45.2+) or Tcra−/− mice were mixed (1:1) with WT NK cells, transferred into Ly49H-deficient hosts (day −1), and then infected with MCMV (day 0). Percentages of transferred Ly49H+ NK cells isolated from WT and Rag1−/− mice (I) and WT and Tcra−/− mice (J) are shown.
Data are representative of two (C, I, and J), three (E and F), and four (A and B) independent experiments. Error bars indicate SEM (n = 3–5). See also Figure S2.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2015 The Authors Terms and Conditions

5. Figure 3
Nod Proteins Expressed by Nonhematopoietic Cells Are Required to Induce KLRG1 Expression on NK Cells
(A) Percentages of KLRG1int+ cells within total NK cell population in the WT mice housed with WT mice (control) or Rag1−/− mice (cohoused) for 3 weeks.
(B) Percentages of KLRG1int+ cells within total NK cell population in the WT mice given control or Abx-treated water for 3 weeks are shown.
(C) Mice were given control (CD45.2+) or Abx-treated water (CD45.1+) for 3 weeks. An equal number of Ly49H+ NK cells isolated from each group was mixed and transferred into Ly49H-deficient mice followed by MCMV infection. Graph shows percentages of each Ly49H+ subset within the total NK cell population.
(D) Percentages of KLRG1int+ cells within the total NK cell population in spleen and peripheral blood from the indicated mice at steady state.
(E–H) Percentage of Ly49H+ NK cells derived from WT (CD45.1+) and Nod1−/− or Nod2−/− (CD45.2+) mice after MCMV infection.
(I) KLRG1− NK cells were sorted from WT (CD45.1+) and Nod2−/− (CD45.2+) mice. An equal number of KLRG1−Ly49H+ NK cells from WT and Nod2−/− mice was mixed (1:1), transferred into Ly49H-deficient hosts (day −1), and then infected with MCMV (day 0). Percentages of transferred Ly49H+ NK cells derived from WT and Nod2−/− mice are shown.
Data are combination (A) or representative (B–I) of two independent experiments. Error bars show SEM (n = 3–5). See also Figure S3.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2015 The Authors Terms and Conditions

6. Figure 4
Commensal Bacteria Are Partially Responsible for Homeostatic Proliferation of NK Cells and Induction of KLRG1 Expression
(A) Enriched WT (CD45.1+) NK cells were labeled with CellTrace Violet, and 5 × 105 cells were transferred via intravenous injection into WT mice (CD45.2+), Tcra−/− mice (CD45.2+), or Rag1−/− mice (CD45.2+) and given either control or Abx-treated water for 14 days prior to transfer.
(B) CellTrace-Violet-labeled WT NK cells were transferred into WT mice (CD45.2+), Rag1−/− mice (CD45.2+), or Rag1−/−Nod2−/− mice (CD45.2+). KLRG1 expression and dilution of CellTrace-Violet in splenic CD45.1+ NK cells were analyzed 7 days after transfer.
Data are representative of three independent experiments with three mice in each experiment. See also Figure S4.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2015 The Authors Terms and Conditions

7. Figure 5 IL-15 Induces KLRG1 Expression
(A) Percentages of KLRG1int+ cells within the total NK cell population from mice with the indicated Il15 genotype.
(B) Percentages of KLRG1int+ cells within the total NK cell population from Rag1−/− mice with the indicated Il15 genotype.
(C–E) Enriched NK cells were labeled with CellTrace Violet, and 5 × 105 cells cell were transferred via intravenous injection into Rag1−/−Il15+/+ mice (CD45.2+) or Rag1−/−Il15+/− mice (CD45.2+) (C); Tcra−/− mice (CD45.2+), Tcra−/− OT-II mice (CD45.2+), or Tcra−/− OT-I mice (CD45.2+) (D); or WT mice (CD45.2+) or Cd28−/− mice (CD45.2+) (E). KLRG1 expression and dilution of CellTrace-Violet in splenic CD45.1+ NK cells were analyzed 7 days after transfer.
(F) Sorted KLRG1− NK cells (4 × 104/well) were cultured in the indicated doses of IL-15 in the presence or absence of T cells (8 × 105/well; a ratio of 20:1 T cells to NK cells to represent relative proportions in the spleen) for 3 days.
(G) KLRG1− NK cells and T cells were cocultured in the same well or in trans well (0.4 μm pore size) cultures in the presence of IL-15 (5 ng/ml) for 3 days. Data are representative of three independent experiments with three mice (C and D), two independent experiments with three mice (E), and triplicates (F and G) per experiment. Error bars show SEM.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2015 The Authors Terms and Conditions

8. Figure 6
Antibiotic Treatment Improves NK Cell Responses to MCMV in T-Cell-Deficient Mice
(A–C) WT, Tcra−/−, and Rag1−/− mice were given control or Abx-treated water for 3 weeks. (A) Percentages of KLRG1int+ cells within the total NK cell population in the indicated mice are indicated. (B) An equal number of Ly49H+ NK cells from Tcra−/− mice (CD45.2+) given control (B) or Abx-treated water (C) were mixed (1:1) with NK cells from WT mice (CD45.1+) and transferred into Ly49H-deficient mice, followed by MCMV infection.
(D and E) An equal number of Ly49H+ NK cells from Rag1−/− mice (CD45.2+) given control (D) or Abx-treated water (E) were mixed with NK cells from WT mice (CD45.1+) and transferred into Ly49H-deficient mice, followed by MCMV infection. Graph shows percentages of each Ly49H+ population within the total NK cell population.
(F–I) Rag1−/− mice were treated with control or Abx-treated water 3 weeks before MCMV infection, and treatment was continued throughout the infection. (F) Number of Ly49H+ NK cells per million peripheral blood leukocytes (red-blood-cells-depleted fraction). (G) Survival of Rag1−/− mice treated with either control water (n = 13) or Abx-treated water (n = 11). (H and I) Copy numbers of MCMV genomic DNA in peripheral blood (H) or oral lavage (I) were quantified by qPCR.
Data are compiled from two independent experiments (G) or representative of two independent experiments with three to five (B–E), five (Abx-treated group in F, H, and I), or seven (control group in F, H, and I) mice per experiment. Error bars show SEM. See also Figure S5.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2015 The Authors Terms and Conditions

9. Figure 7
Reciprocal Regulation of Memory NK Cell Progenitors by Commensal Bacteria and T Cells—A Hypothetical Model
Nod receptors on host nonhematopoietic cells sense commensal bacteria and enhance IL-15 production in myeloid cells. Competitive consumption of IL-15 by T cells inhibits the induction of KLRG1int+ NK cells and preserves progenitor KLRG1− NK cells that preferentially develop into KLRG1high memory NK cells.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2015 The Authors Terms and Conditions